LRRK2 phosphorylates novel tau epitopes and promotes tauopathy

Anti-LRRK2 rabbit polyclonal antibody (1182) was previously described [72]. MJFF-4 (c81-8) was obtained from the Michael J. Fox Foundation. Anti-pT149 and anti-pT153 tau specific antibodies were made as a service by GenScript USA Inc. Briefly, rabbits were immunized with the pT149 peptide (DGKpTKIATPRGAAC) or pT153 peptide (DGKTKIApTPRGAAC), affinity purified with the same peptide, and negatively absorbed against the non-phosphorylated peptide (DGKTKIATPRGAAC). We used polyclonal tau antibodies E1 (specific for amino acids 19–33 of human tau) [11], pT205 (Abcam), pT212 (Anaspec), pS214 (Invitrogen) [70], 17025 anti-tau (provided by Dr. Virginia Lee, University of Pennsylvania, Philadelphia, PA); and monoclonal tau antibodies CP13 (specific for pS202 in tau), PHF1 (specific for pS396/S404 in tau), and MC1 [pre-PHF conformational epitope (7–9 and 326–330)] (provided by Dr. Peter Davies, The Einstein Institute for Medical Research, Manhasset, NY, USA), AT8 (specific for pS199/pS202/pT205 in tau), AT100 (specific for pT212/S214 in tau), and AT270 (specific for pT181 in tau) (Innogenetics, Fisher Scientific). Other monoclonal antibodies used were anti-V5 (Invitrogen) and anti-GAPDH (Biodesign).

Unless otherwise noted, kinase assays were set up in a total volume of 25 μl with 25 nM recombinant GST–LRRK2 (970–2,527) in 50 mM Tris/HCl (pH 7.5), 0.1 mM EGTA, 10 mM MgCl₂ and 0.2 mM [γ-³²P] ATP (4 Ci/mmol) in the presence of 10 μM of tau or MBP substrate, unless otherwise noted. After incubation for 60 min at 30 °C, reactions were terminated by applying to individual 2.5 cm-diameter disks of P-81 phosphocellulose filter paper (Schleicher & Schuell, Keene, NH) that were immediately immersed in 75 mM phosphoric acid. After extensive washing with 75 mM phosphoric acid, P-81 filters were rinsed with acetone and allowed to air dry. Filters were immersed in Cytoscint liquid scintillation cocktail (Fisher Scientific) and ³²P radioactivity on each filter was measured by liquid scintillation using an LS6500 counter (Beckman Coulter). K _m and V _max parameters were calculated using Graph-Pad Prism v5.02 (GraphPad Software).

For phosphorylation analysis of recombinant tau proteins with LRRK2, reactions were stopped with the addition of SDS-sample buffer and heating to 100 °C for 5 min. Samples were resolved onto SDS-polyacrylamide gels, and incorporation of phosphate was determined by autoradiography and/or immunoblotting with phospho-specific antibodies.

As described above, 10 μM of 0N3R tau was subjected to overnight in vitro phosphorylation by either G2019S LRRK2 or the kinase dead (KD) LRRK2 mutant D1994K. Samples were resolved onto SDS-polyacrylamide gels, and then excised and washed in 50 % acetonitrile and water [5]. To maximize sequence coverage, three equivalently loaded and excised gel bands of 0N3R tau were separately digested with trypsin, chymotrypsin or GluC. Mass spectrometry (MS) analysis of modified 0N3R tau was performed as a service by the Harvard Mass Spectrometry and Proteomics Resource Laboratory (Cambridge, MA, USA). Peptide sequence analysis of each digestion mixture was performed by microcapillary reversed-phase high-performance liquid chromatography coupled with nanoelectrospray tandem mass spectrometry (μLC–MS/MS) on an LTQ-Orbitrap Velos mass spectrometer (ThermoFisher Scientific, San Jose, CA, USA). The Orbitrap repetitively surveyed an m/z range from 395 to 1,600, while data-dependent MS/MS spectra on the twenty most abundant ions in each survey scan were acquired in the linear ion trap. MS/MS spectra were acquired with relative collision energy of 30 %, 2.5-Da isolation width, and recurring ions dynamically excluded for 60 s. Preliminary sequencing of peptides was facilitated with the SEQUEST algorithm [15] with a 30 ppm mass tolerance against the human subset of the Uniprot Knowledgebase supplemented with a database of common laboratory contaminants, concatenated to a reverse decoy database. Using a custom version of Proteomics Browser Suite (PBS v.2.7, ThermoFisher Scientific, San Jose, CA, USA) peptide-spectrum matches (PSMs) were accepted with mass error <2.5 ppm and score thresholds to attain an estimated false discovery rate of ~1 %. Data-sets for all digest results were combined in silico, culled of minor contaminant PSMs, and re-searched with SEQUEST algorithm against the 0N3R tau sequence without taking into account enzyme specificity and with differential modifications of phosphorylated tyrosine, serine, and threonine residues. The discovery of phosphopeptides and subsequent manual confirmation of their MS/MS spectra were facilitated using in-house versions of programs MuQuest, GraphMod, and FuzzyIons (PBS, ThermoFisher Scientific).

Recombinant C-terminal fragment of human 3R tau [C′ Tau] corresponding to amino acids 244–441 minus amino acids 275–305 that would be present in 2N4R tau human tau was phosphorylated with recombinant glycogen synthase kinase 3 beta (GSK-3β) (New England BioLabs Inc, Ipswich, MA, USA) (20 mM Tris, pH 7.5, 10 mM MgCl₂, 5 mM DTT, 200 μM ATP, 1 mg/ml tau and 20,000 units enzyme in 50 μl) for 1 h at 30 °C. Controls included similar reactions without the kinase. 100 ng of each synthetic peptide or 500 ng of tau recombinant protein diluted in 100 μl of water was absorbed per well of 96-well EIA/RIA Corning plates (Corning, NY, USA). The plates were extensively washed with PBS and blocked with PBS/5 % fetal bovine serum (FBS). Primary antibodies as indicated were diluted in PBS/5 % FBS and incubated on the plates. After extensive washes with PBS, plates were incubated with either anti-mouse antibody conjugated to HRP or anti-rabbit antibody conjugated to HRP diluted in PBS/5 % FBS. Plates were again extensively washed with PBS and reactions were carried out with 3,3′,5,5′-tetramethylbenzidine (TMS) reagents (KPL, Gaithersburg, NY, USA). The reactions were terminated by adding 0.2 M HCl and optical density was measured at OD₄₅₀ using a Multiskan Plus plate reader (ThermoFisher). Experiments were performed in quadruplicate.

Mice were housed and treated in accordance with the NIH Guide for the Care and Use of Laboratory Animals. All animal procedures were approved and conducted in accordance with the Mayo Clinic Institutional Animal Care and Use committee and the University of Florida Institutional Animal Care and Use committee. Mice were maintained in a pathogen-free facility on a 12 h light/dark cycle with water and food provided ad libitum.

The parental Tau_P301L responder line and parental tTA activator line were generated and maintained on an FVB and 129/S6 background, respectively, as previously described [65]. Bigenic rTg4510 mice have forebrain-focused expression of P301L transgenic tau. The parental bacterial artificial chromosome (BAC)-LRRK2 mice, maintained on an FVB background, contain the entire human LRRK2 gene including regulatory sequences and LRRK2 expression is driven by the human LRRK2 promoter, as previously described [46]. Mice from the Tau_P301L responder line were crossed with mice from the BAC-LRRK2 mouse line for one generation to obtain LRRK2.Tau_P301L responder mice on an FVB background. LRRK2.Tau_P301L responder mice were then crossed with mice from the tTA activator line to obtain the resultant F1 LRRK2/Tau_P301L mice on a 50 % FVB, 50 % 129S background, the same as the original rTg4510 mouse model [65]. All mice in this study were harvested at 5.5 months of age when mature cortical tangles and hippocampal neurodegeneration are detectable in Tau_P301L only animals. We harvested 10 mice per group, half male and half female, for the four genotypes of interest (non-transgenic, LRRK2, Tau_P301L, and LRRK2/Tau_P301L). One male LRRK2/Tau_P301L mouse did not overexpress LRRK2 (as determined by western blotting) and was excluded from all studies. The sarkosyl-preparation (see protocol below) of one female Tau_P301L mouse had extremely high tau aggregation via western blotting and was identified by Grubb’s analysis to be an outlier. As this may have been a tissue preparation error, all western blot data from this mouse were excluded from the final results.

All mice were euthanized by cervical dislocation to maintain the brain biochemistry by avoiding anesthesia-induced tau changes. Brains were quickly removed, cut down the midline, and one brain half was drop fixed in 10 % formalin for 24 h for immunohistochemical analysis and the other brain half was immediately homogenized to preserve the LRRK2 protein. Brains were homogenized in 6 volumes of homogenate buffer [50 mM Tris–HCl, pH 8.0, 274 mM NaCl, 5 mM KCl, 1 % protease inhibitor mixture (Sigma), 1 % phosphatase inhibitor cocktails I and II (Sigma), and 1 mM phenylmethylsulfonyl fluoride (PMSF)]. For LRRK2 analysis, homogenates were centrifuged at 150,000×g for 15 min at 4 °C. The supernatants were collected and protein concentration was determined using BCA protein assay reagent and BSA as the standard. For tau analysis, the brain homogenate was diluted to 10 volumes using Tris-buffered saline and subjected to sarkosyl fractionation as previously described [65]. Specifically, 200 μl of 10× homogenate was centrifuged at 150,000×g for 15 min at 4 °C and the supernatant, which contains soluble tau species, was collected and protein concentration was determined as described above. Pellets were homogenized in 3 volumes (600 μl) of Buffer B [10 mM Tris–HCl (pH 7.4), 0.8 M NaCl, 10 % Sucrose, 1 mM EGTA and 1 mM PMSF] and centrifuged at 150,000×g for 15 min at 4 °C. The supernatants were collected and incubated with 1 % sarkosyl (Sigma) for 1 h at 37 °C, followed by centrifugation at 150,000×g for 30 min at 4 °C to obtain a sarkosyl-soluble supernatant and sarkosyl-insoluble pellet. The sarkosyl-insoluble pellet, which contains the biochemical equivalent of neurofibrillary tangles, was re-suspended in 20 μl TE buffer [10 mM Tris–HCl (pH 8.0), 1 mM EDTA].

For in vitro and cell culture experiments, equal amounts of protein samples were loaded and resolved by SDS-PAGE, followed by electrophoretic transfer onto nitrocellulose membranes. Membranes were blocked in Tris-buffered saline (TBS) with 5 % non-fat milk powder, and incubated overnight with 1182 LRRK2-specific antibody [72], anti-V5 antibody (Invitrogen), or anti-tau antibody (17025) in TBS/5 % non-fat milk powder. Membranes were also incubated overnight with pT149 or pT153 tau specific antibodies in TBS/5 % BSA. Each incubation was followed by goat anti-mouse conjugated horseradish peroxidase (HRP) (Amersham Biosciences) or goat anti-rabbit HRP (Cell Signaling Technology), and immunoreactivity was detected using chemiluminescent reagent (NEN) followed by exposure on X-ray film.

For analysis of LRRK2 protein in mouse brain tissue, 50 μg of protein was diluted in NuPAGE LDS-sample buffer with NuPAGE reducing agent (Invitrogen), boiled for 3 min at 95 °C, loaded onto 26-well 3–8 % Tris–Acetate gels (Invitrogen), separated by SDS-PAGE and transferred in NuPAGE transfer buffer (Invitrogen) to PVDF membranes (Millipore, Fisher Scientific). Membranes were blocked for 1 h at room temperature in 5 % non-fat milk powder in TBS with 0.1 % Triton X-100 (TBS-T) and then incubated overnight in primary antibody diluted in 5 % non-fat milk powder in TBS-T at 4 °C. For tau protein analysis from mouse brain, 5 μg of the soluble fraction or 3 μl of the sarkosyl-insoluble fraction was diluted in Novex Tris–glycine SDS-sample buffer (Invitrogen) with β-mercaptoethanol, heat denatured at 95 °C for 5 min, loaded onto 26-well 10 % Tris–glycine gels (Invitrogen), separated by SDS-PAGE and transferred in CAPS transfer buffer (Sigma) to PVDF membranes. Membranes were blocked in TBS-T with 5 % non-fat milk powder and incubated overnight with antibody in TBS-T/5 % non-fat milk powder, except for pT149 and pT153 tau antibodies, which were in TBS-T/5 % BSA. All membranes were washed 3 times in TBS-T, incubated with either goat anti-mouse HRP or goat anti-rabbit HRP secondary antibodies (Jackson ImmunoResearch) for 1 h at room temperature and washed again 3 times in TBS-T. Membranes were developed using Western Lightning Plus (Perkin Elmer) and imaged using a FluorChem E System (ProteinSimple). The relative levels of immunoreactivity were determined by densitometry using the software AlphaView SA (ProteinSimple).

Fixed mouse brains were paraffin embedded and cut into 5 μm sagittal sections. Hematoxylin and eosin (H&E) staining was performed on at least two brain sections from each mouse to align all brains to approximately 1.3 mm lateral to the midline using a mouse brain atlas [52]. Immunohistochemistry with the Dako Universal Autostainer with DAKO Envision + HRP system (Dako) was performed with the following antibodies: pT149 tau, pT153 tau, AT8, AT270, CP13, and MC1. Stained slides were digitally scanned using a ScanScope XT scanner and were analyzed using ImageScope version 11.2.0.780 software (Aperio). Positive pixel count algorithms were created to measure the staining density of the secondary antibody, specifically chromogen 3,3′ diaminobenzidine. The cortex of each animal was traced and analyzed using these algorithms and the burden was expressed as a percentage of immunostained pixels to total area. Sections that had tears or other artifacts were not included in the analysis.

Paraffin embedded sections were processed for immunohistochemistry as above for mice from 3R + 4R tauopathy (5 AD—average age: 90 ± 6 years; 4 women; median Braak NFT Stage: VI; 2 with concurrent diffuse cortical Lewy bodies), 4R tauopathy (4 PSP and 1 CBD—average age: 77 ± 7 years; 4 women; median Braak NFT Stage: III), 3R tauopathy (2 PiD—average age: 68 ± 6 years; 1 woman; median Braak NFT Stage: III), and mutant LRRK2 carriers (4 G2019S—average age: 76 ± 5 years; 2 women; Braak NFT Stage: 0–V). Sections were counterstained with hematoxylin.

Grubb’s analysis was used to identify outliers in the western blot analysis of sarkosyl-insoluble tau. Data are presented as the mean ± standard error mean unless otherwise noted. Analysis of two groups was performed with an unpaired, two-tailed Student’s t test while analysis of multiple groups was analyzed by one-way ANOVA and post hoc Bonferroni multiple comparisons test (α = 0.05). Western blot and immunohistochemical data from male and female Tau_P301L and LRRK2/Tau_P301L mice were analyzed by two-way ANOVA with genotype and sex as independent variables. All analyses were performed using GraphPad Prism version 6.00 software (GraphPad Software) and in all cases, P ≤ 0.05 was considered to be statistically significant.

To determine if LRRK2 directly phosphorylates tau, recombinant tau (2N4R) was incubated with multiple forms of recombinant GST-LRRK2 (970–2,527) and incorporation of γ-[³²P] ATP was determined by autoradiography. Both wild-type (WT) and mutant (G2019S, I2020T and R1441C) LRRK2 phosphorylated recombinant tau in vitro (Fig. 1a). Although we analyzed three different LRRK2 mutants, each of which has been implicated in familial PD with neurofibrillary tangle pathology, G2019S LRRK2 yielded the highest level of tau phosphorylation. Further, tau phosphorylation was largely ablated when using the LRRK2 kinase dead/impaired (KD) D1994K mutant (Fig. 1a), indicating that LRRK2 directly phosphorylated tau in vitro. We then examined the ability of mutant LRRK2 to phosphorylate different tau isoforms that contain alternatively spliced N-terminal (0, 1 or 2 N) and C-terminal MT-binding domains (3R or 4R). Recombinant GST-G2019S LRRK2 (970–2,527) phosphorylated all forms of tau regardless of isoform or the presence of FTDP-17t-associated mutations (E342V, P301L, P301S and R406W) (Fig. 1b). These findings were confirmed using full-length G2019S LRRK2 (Supplemental Fig. 1).

To identify the tau residues that are phosphorylated by LRRK2 in vitro, we performed MS analyses of tau (0N3R) phosphorylated with GST-G2019S LRRK2 (970–2,527). Using the shortest tau isoform with the highest kinase active LRRK2, we had the greatest likelihood of complete coverage. We obtained >99 % sequence coverage from the 0N3R isoform and identified multiple tau residues phosphorylated by G2019S LRRK2 (Fig. 2a), with T149, T153, T169, T205, T263 and T231 identified as the major phosphorylation sites by LRRK2 (Supplemental Table 1). These modifications were not present in identical MS analysis of 0N3R tau incubated with KD LRRK2. To parse the sites that were preferentially phosphorylated by LRRK2, we incubated various forms of recombinant LRRK2 with synthetic peptides that spanned short regions of tau containing 1–3 potential tau phospho-epitopes (Supplemental Table 2) and quantified the resultant phosphorylation. G2019S-LRRK2 showed the greatest phosphorylation of the Tau-A (KKAKGADGKTKIATPRGAAPPGQK) and Tau-B (REPKKVAVVRTPPKSPSSAKSRL) peptides, with the WT Tau-B peptide phosphorylated 83 ± 1.7 % (P < 0.001) less than the WT Tau-A peptide (Fig. 2b) as assessed by one-way ANOVA and post hoc Bonferroni multiple comparisons test. The other peptides demonstrated minimal phosphorylation. These data were consistent with the MS data, which showed that pT149 and pT153 peptides were among the most abundant phosphorylated products resulting from LRRK2-mediated tau phosphorylation. Alanine substitution to block site-specific LRRK2 phosphorylation of the Tau-A peptide reduced phosphorylation by 93 ± 1.2 % (T149A; P < 0.0001) and 21 ± 1.2 % (T153A; P < 0.001) compared to WT Tau-A (Fig. 2b), indicating that tau T149 and T153 were primary and secondary targets, respectively, of LRRK2-directed phosphorylation. We then assessed the kinetic features of WT and G2019S LRRK2 phosphorylation of Tau-A (see “Materials and methods”) where the concentration of Tau-A was varied as indicated while the concentration of ATP was constant at 200 μM. The K _m of WT and G2019S LRRK2 for the Tau-A peptide was determined to be ~170 μM, which is similar to that of LRRKtide, the most robust and commonly used LRRK2 substrate in kinetic studies [1, 9, 31, 48]. Furthermore, like LRRKtide, the V _max of tau phosphorylation by G2019S LRRK2 was ~threefold greater than that of WT LRRK2 (Fig. 2c) [9].

To assess phosphorylation of tau T149 or T153, we created antibodies specific for each phospho-epitope (see “Materials and methods”). ELISA analysis of pT149 and pT153 tau antibodies was used to establish the phospho-specificity of each antibody. Results showed that both antibodies are highly specific for tau peptides phosphorylated at their respective epitopes with minimal reactivity to non-phosphorylated tau peptide, non-phosphorylated C-terminal tau [C′ Tau] and C′ Tau phosphorylated by GSK-3β (Fig. 3a). In addition, pT149 antibody had minimal cross reactivity to tau peptide phosphorylated at T153 and the pT153 antibody had low cross reactivity to tau peptide phosphorylated at T149, but none when compared to the other control peptides. Both pT149 and pT153 antibodies recognized tau that had been incubated with GST-G2019S LRRK2 (970–2,527), but not with GST-KD LRRK2 (970–2,527) (Fig. 3b). Further, HEK 293T cells were co-transfected with expression plasmids for 2N4R tau and pcDNA3.1, untagged full-length WT or G2019S LRRK2, or V5-tagged full-length WT or G2019S LRRK2. Immunoblot analysis of cell lysates with total tau and pT149 tau antibodies confirmed that both tagged and untagged full-length WT and G2019S LRRK2 increased phosphorylation of tau at T149 in vivo by 8.4 ± 0.8-fold more (WT; P < 0.0001) and 7.5 ± 0.4-fold more (G2019S; P < 0.0001) than vector transfected control cells when normalized to total tau (Fig. 3c).

To test the pathological relevance of pT149 and pT153, we performed immunohistochemistry on brain tissue from different human tauopathies using our pT149 and pT153 tau antibodies. Both antibodies recognized neurofibrillary pathology in AD (Fig. 3d, left panels), including neurofibrillary tangles, neuropil threads and dystrophic neurites in senile plaques. Further, both antibodies stained neurofibrillary tangles and tufted astrocytes (Fig. 3d, middle-left panels) in PSP, as well as Pick bodies in PiD (Fig. 3d, middle-right panels). Moreover, the pT149 and pT153 antibodies stained pathological tau in patients with the G2019S LRRK2 mutation (Fig. 3d, right panels). In addition to the tangle pathology previously reported for G2019S mutant carriers, our antibodies identified tau surrounding Lewy bodies in the brainstem (Fig. 3e). These results indicate that in addition to G2019S-LRRK2 carriers, tau hyperphosphorylation at T149 and T153 occurs in a range of human tauopathies, including so-called 4R-tauopathies, 3R-tauopathies and 3R + 4R tauopathies, and that phosphorylation of these epitopes is linked to pathology, since no staining was detected in unaffected brains or unaffected brain regions in tauopathies (data not shown).

To determine, if LRRK2 could modify the development of tauopathy in vivo, we crossed human WT LRRK2 BAC mice that do not have an overt phenotype or pathological abnormalities (including evidence of tau pathology) [46], with rTg4510 mice that express human mutant (P301L) 0N4R tau [65] to generate mice that expressed both transgenes (LRRK2/Tau_P301L) (see breeding scheme in Supplemental Fig. 2). LRRK2 BAC mice showed the highest expression of transgenic LRRK2 in the hippocampus as well as high expression in the cortex [46]. These regions overlap well with the forebrain-focused transgenic tau expression that is conditionally driven by a CAMKIIα-tTA transgene in the rTg4510 mice [65], reducing the likelihood that an interaction between the proteins would be missed due to disparate expression profiles. The forebrain of 5.5-month-old rTg4510 mice, hereafter termed Tau_P301L, has been shown previously to have insoluble hyperphosphorylated tau, neurofibrillary tangles, and neuronal loss [59, 65]. We, therefore, examined LRRK2/Tau_P301L mice at 5.5 months of age for alterations in this well-characterized pathology.

Initially, we confirmed that all LRRK2/Tau_P301L experimental mice expressed the LRRK2 (Fig. 4a, b) and tau (Fig. 4c, d) transgenes. Soluble tau fractions typically contain normal tau species that run at ~55 kDa which can be used to determine equal levels of expression across mice. Human LRRK2 expression in LRRK2/Tau_P301L mice did not affect human tau levels in the normal (soluble) fraction (Fig. 4c, d) when compared to Tau_P301L mice alone, ensuring that any effects on tauopathy in the LRRK2/Tau_P301L mice would not be due to expression artifact. With a panel of phospho-specific tau antibodies, we then analyzed soluble tau via western blotting for phosphorylation at sites that we confirmed to be directly phosphorylated by LRRK2 in vitro (T149 and T153) and/or identified by our initial MS results (T181, T212 and T205). We also included epitopes that are associated with human disease and were not identified by our MS analysis (S202, S214, and S396/S404). We did not detect any changes in the phosphorylation of the ~55 kDa soluble tau in LRRK2/Tau_P301L mice when compared to the Tau_P301L mice with any antibody utilized (Supplemental Fig. 3). We and others have previously reported that species of higher molecular weight tau can be observed in the soluble fraction, potentially representing some species of aggregated tau [39, 63, 64]. Interestingly, our pT153 antibody recognized a ~64 kDa band in the soluble fraction of both Tau_P301L and LRRK2/Tau_P301L mice (Supplemental Fig. 3a). Comparison of this band between both cohorts (P = 0.17) failed, however, to reach our 0.05 threshold for significance.

An important feature of human tauopathy and a subset of mutant LRRK2 carriers is the aggregation of tau. In tauopathies, tau aggregation is associated with the shift of tau into the biochemically abnormal, sarkosyl-insoluble fraction. We, therefore, examined the sarkosyl-insoluble fractions of brains from LRRK2/Tau_P301L mice by western blot analysis to determine if LRRK2 enhances tau aggregation compared to Tau_P301L mice. Using the phospho-independent, human tau antibody, E1, we found that insoluble, aggregated tau levels were ~3.5 times higher in LRRK2/Tau_P301L compared to Tau_P301L mice (Fig. 5a, b). Insoluble tau from both LRRK2/Tau_P301L and Tau_P301L mice migrated primarily as a ~64 kDa band—a species that we have previously shown to correlate with the presence of NFTs in tau transgenic mice [39]. Using a two-way ANOVA (genotype × sex), we then determined that the levels of sarkosyl-insoluble human tau in Tau_P301L and LRRK2/Tau_P301L mice were affected by both genotype [F(1, 14) = 34, P < 0.0001] and sex [F(1, 14) = 34, P < 0.0001], and that there was also a significant interaction between both factors [F(1, 14) = 14, P < 0.01]. To help interpret these findings, we analyzed females and males separately and found that female LRRK2/Tau_P301L mice had approximately 3.6 times more insoluble tau compared to female Tau_P301L mice (P < 0.001) (Supplemental Fig. 4a), and that male LRRK2/Tau_P301L mice had 2.8 times more insoluble tau compare to male Tau_P301L mice (P < 0.05) (Supplemental Fig. 4b).

We then analyzed the sarkosyl-insoluble fraction of Tau_P301L and LRRK2/Tau_P301L mouse brains using our panel of phospho-tau antibodies and found that LRRK2 increased phosphorylation of insoluble tau at all epitopes that were examined (Supplemental Figs. 5 and 6). To determine if the elevated levels of phosphorylated tau were simply driven by the overall increase of aggregated tau in the insoluble fraction, we normalized the densitometry of each phospho-tau antibody by the densitometry of sarkosyl-insoluble human tau for each mouse. When normalized for the amount of insoluble tau in the sarkosyl-insoluble fraction, analysis by two-way ANOVA (genotype × sex) revealed a main genotype effect with increased phosphorylation of T149, T205, and S199/S202/T205 (Fig. 5c, g, h; Table 1) and decreased phosphorylation of T181 in LRRK2/Tau_P301L mice compared to Tau_P301L mice (Fig. 5e; Table 1). Phosphorylation of T153 in LRRK2/Tau_P301L mice compared to Tau_P301L mice (P = 0.07) did not reach our significance threshold (Fig. 5d; Table 1) and phosphorylation of S202, T212, S214, T212/S214 and S396/S404 was unchanged by co-expression of LRRK2 in Tau_P301L mice (S202: P = 0.62; T212: P = 0.32; S214: P = 0.90; T212/S214: P = 0.63; and S396/S404: P = 0.40) (Fig. 5f, i–l; Table 1). In addition, this analysis revealed a main sex effect for pT181 (AT270), S199/S202/T205 (AT8) and S396/S404 (PHF1), but not the other epitopes examined (see statistical summary in Table 1).

We sought to validate our biochemical studies with neuropathological analysis of tau burden using antibodies that detect specific phosphorylation or conformational tau epitopes. All antibodies identified prominent tau pathology, including neurofibrillary tangles, in the brains of Tau_P301L and LRRK2/Tau_P301L mice, which was absent in the LRRK2 only transgenic and non-transgenic (nTg) mice (Fig. 6). Tau pathology was distributed throughout the cortex and hippocampus of Tau_P301L and LRRK2/Tau_P301L mice; however, we focused our assessment on the cortex since neuronal loss within the hippocampus can confound accurate assessment of tau burden at this age. Using two-way ANOVA analysis, we observed significant increases in phosphorylated tau pathology in LRRK2/Tau_P301L mice with pT149, pT153, CP13, and AT8 immunostaining when compared to Tau_P301L mice (Figs. 6, 7; Table 1). Although the burden of tau pathology with these antibodies was also significantly influenced by sex, where females had greater burden than males, there was no interaction between the genotype and sex (Fig. 7; Table 1). In addition, we observed significant increases in conformationally-abnormal tau in LRRK2/Tau_P301L mice with MC1 immunostaining when compared to Tau_P301L mice (Figs. 6, 7; Table 1). Tau burden as assessed with MC1 was also significantly influenced by sex, but there was no interaction between genotype and sex (Table 1). No significant difference in tau burden was observed with AT270, irrespective of genotype or sex (Figs. 6, 7; Table 1).